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A simple cloning-free method to efficiently induce gene expression using CRISPR/Cas9

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Fang, L, Hung, SSC, Yek, J, El Wazan, L, Nguyen, T, Khan, S, Lim, SY, Hewitt, AW ORCID: 0000-0002-5123-5999 and Wong, RCB 2019 , 'A simple cloning-free method to efficiently induce gene expression using CRISPR/Cas9' , Molecular Therapy - Nucleic Acids, vol. 14 , pp. 184-191 , doi: 10.1016/j.omtn.2018.11.008.

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Abstract

Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here, we report a simple, cloning-free method to induce gene expression at an endogenous locus using CRISPR/Cas9 activators. Our strategy utilizes synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65, and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1, and NEUROD1) in human cells, whereas the rat cells exhibit more variable and less-efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applied as a rapid workflow to initiate gain-of-function studies for a range of molecular- and cell-biology disciplines.

Item Type: Article
Authors/Creators:Fang, L and Hung, SSC and Yek, J and El Wazan, L and Nguyen, T and Khan, S and Lim, SY and Hewitt, AW and Wong, RCB
Keywords: CRISPR, CRISPR activation, SaCas9, SpCas9, cloning free, gene activation, multiplex gene activation, transcriptional activator
Journal or Publication Title: Molecular Therapy - Nucleic Acids
Publisher: Nature Publishing Group
ISSN: 2162-2531
DOI / ID Number: 10.1016/j.omtn.2018.11.008
Copyright Information:

Copyright 2018 The Authors. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) https://creativecommons.org/licenses/by-nc-nd/4.0/

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